Related Experiment Video
Updated: Feb 6, 2026
Purification of Ubiquitinated p53 Proteins from Mammalian Cells
Published on: March 21, 2022
The Impact of p53 Dysfunction in ATR Inhibitor Cytotoxicity and Chemo- and Radiosensitisation
Fiona K Middleton1, John R Pollard2, Nicola J Curtin3
1Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne NE2 4HH, UK. fmiddleton@cancertechnology.com.
Abstract:
Ataxia telangiectasia mutated and Rad3 related kinase (ATR) signals replication stress and DNA damage to S and G2 arrest and promotes DNA repair. Mutations in p53, critical for G1 checkpoint control, are common in cancer and predicted to confer vulnerability to ATR inhibitors. Reported data on the impact of p53 status are variable possibly because of the use of unmatched cells and surrogate endpoints of survival. The cytotoxicity of VE-821 alone and its ability to potentiate radiation and gemcitabine cytotoxicity was determined in isogenic and unmatched p53 wild-type (wt) and null/mutant cells, as well as immortalised nonmalignant MCF10 (immortalised non-neoplastic) cells, by colony-forming assay. The effect on cell cycle checkpoints was determined by flow cytometry. The isogenic p53 defective cells were not more sensitive to VE-821 alone. Defective p53 consistently conferred greater chemo- and radiosensitisation, particularly at high dose levels in isogenic cells but not unmatched cells. VE-821 did not sensitise MCF10 cells. We conclude that p53 status is just one factor contributing to chemo- and radiosensitisation by ATR inhibition, the lack of chemo- or radiosensitisation in the noncancerous cells suggests an element of tumour-specificity that warrants further investigation. The greater sensitisation at high-dose irradiation suggests that ATR inhibitors may be most effective with hypofractionated radiotherapy.
Insights
Ataxia telangiectasia mutated and Rad3 related kinase (ATR) inhibitors show promise in cancer therapy. p53 status influences chemo- and radiosensitization, with potential efficacy in hypofractionated radiotherapy.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- Ataxia telangiectasia mutated and Rad3 related kinase (ATR) is crucial for signaling replication stress and DNA damage, arresting cells in S and G2 phases to promote repair.
- Mutations in p53, essential for G1 checkpoint control, are prevalent in cancers and theoretically increase vulnerability to ATR inhibitors.
- Existing data on p53's impact on ATR inhibitor efficacy are inconsistent, potentially due to the use of unmatched cells and surrogate survival endpoints.
Purpose of the Study:
- To investigate the cytotoxicity of the ATR inhibitor VE-821 alone and in combination with radiation and gemcitabine.
- To determine the influence of p53 status (wild-type vs. null/mutant) on cellular sensitivity to VE-821, chemotherapy, and radiotherapy.
- To assess the effect of ATR inhibition on cell cycle checkpoints and explore potential tumor-specific effects.
Main Methods:
- Cytotoxicity was evaluated using colony-forming assays in isogenic and unmatched p53 wild-type and null/mutant cancer cells, as well as immortalized non-neoplastic MCF10 cells.
- Cell cycle checkpoint modulation was analyzed via flow cytometry.
- The potentiation of radiation and gemcitabine cytotoxicity by VE-821 was assessed.
Main Results:
- Isogenic p53-defective cells did not exhibit increased sensitivity to VE-821 monotherapy.
- Defective p53 status consistently led to greater chemo- and radiosensitization, particularly at high doses in isogenic cells, but this was not observed in unmatched cells.
- VE-821 did not sensitize non-neoplastic MCF10 cells, suggesting potential tumor-specific effects.
- ATR inhibition showed greater sensitization with high-dose irradiation.
Conclusions:
- p53 status is one of several factors influencing chemo- and radiosensitization by ATR inhibition.
- The lack of sensitization in non-cancerous cells indicates a potential tumor-specific therapeutic window for ATR inhibitors.
- ATR inhibitors may be most effective when combined with hypofractionated radiotherapy due to enhanced sensitization at high radiation doses.
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